The long term objective of the experiments is to gain an understanding at the molecular level of how proteins become localized to mitochondria in eukaryotic cells. In particular a detailed genetic analysis of import of the yeast HEM1 product, Delta-amino levalinate (ALA) synthase, will be conducted. Mutations resulting in single amino acid changes or short substitutions in the ALA synthase signal sequence will be isolated by a combined approach of gene fusion, in vitro mutagenesis and genetic selection. After crossing these mutations into the yeast genome generating Hem- mutants, suppressors that recognize the altered signals will be selected as Hem+ revertants. In a separate selection, mutants with a reduced ability to recognize the wild-type ALA synthase signal will be isolated. A genetic analysis of all mutations isolated will be carried out. Whether import of other mitochondrial proteins is altered in mutant strains will be tested. Effects of mutations on import in vitro will be determined to pinpoint whether a component of the import machinery has been mutated, and, if so, whether that component is cytosolic or mitochondrial. Genes that are believed to encode components of the import machinery will be cloned, sequenced, and fused to lacZ. Antibody raised to the fusions will be used to determine the location of the products of cloned genes in yeast cells. Selected proteins will be overexpressed, purified, and employed in biochemical studies. Finally, the role played by mitochondrial signal sequences in regulating import of two other yeast proteins, citrate synthase and cytochrome oxidase subunit IV, will be analyzed.